Chemical reaction apparatus for performing multiple reaction on a surface and collecting the product

ABSTRACT

Chemical reaction apparatus, materials and methods are provided for the automatable, efficient synthesis of chemical species and libraries. In accordance with preferred embodiments, chemical droplet generation and direction techniques are employed to prepare oligomers and libraries of chemical species. Reaction assemblies adapted for efficient synthetic employment and for improved collection are also disclosed.

This is a division, of application Ser. No. 08/309,925, filed Sep. 21,1994, the disclosure of which is herein incorporated by reference.

FIELD OF THE INVENTION

This invention is concerned with novel apparatus, materials andmethodologies for synthesizing chemical compounds, especially oligomers.Chemical reactions are accomplished on surfaces in a fashion which isboth easy and economical and which is amenable to the attainment of highyields. Automation of chemical reaction processes is facilitated in thepresent invention. Synthesis of oligomers, especially oligonucleotidesand polypeptides, is especially benefitted by the employment of thepresent invention. A wide variety of other chemical reactions can beachieved in accordance with the present invention, however. The presentinvention is also suited for the preparation of chemical libraries whichare useful per se, inter alia for screening purposes and otherwise.

BACKGROUND OF THE INVENTION

It has been proposed heretofore to employ apparatus commonly called an"ink jet" for the delivery of deblocking reagents in solid stateoligomeric reactions. Such proposal, however, is crude, is limited inscope, and generally requires non-automatable procedures for itsemployment. Thus, it has been proposed to use an "ink jet" apparatus toplace droplets of a deblocking reagent, zinc bromide, upon specifiedlocations of a reaction surface to deblock a growing oligonucleotidechain to render it amenable to chain elongation. This has been proposedfor use, for example, on a microscope slide. Following the delivery ofdeblocking reagent, it was proposed to manipulate the microscope slidesuch as by dipping it into a quantity of further reagent to accomplish achain elongation. Further application of the "ink jet" delivery ofchemical reagent was then proposed, however realignment of themicroscope slide would be necessitated by that proposed methodology. Inaddition to the cumbersome nature of the prior proposal and its lack ofsuitability to full automation, only relatively small harvests ofoligomeric product were anticipated using the proposed scheme.

There is a great need for chemical reaction apparatus, materials andattendant methodologies which permit the automated, high yield,relatively large scale synthesis of chemical species, especiallyoligomers. The apparatus and methodologies provided by the presentinvention now enable the use of chemical "jetting" technology for thepractical synthesis of chemical and biochemical products in high yieldand with ease of synthesis. There is also provided a need for syntheticsystems which deliver complex synthesized products to receiving vesselswith ease and in high yield.

The apparatus and methods of the present invention also address along-felt need by permitting the preparation of libraries of chemicalcompounds having predictable diversity among the functional moietiesthereupon.

This invention also diminishes waste stream pollution associate withmany prior synthetic technologies through the precision application ofreagent moieties in synthetic schemes.

Precisely arrayed pluralities of defined chemical compounds are alsopossible through employment of this invention. Binding, reaction,degradation, chemical and biological interaction and other testingprotocols may, thus, be performed with unparalleled convenience throughpractice of embodiments of this invention.

The invention minimizes reagent usage such that the impact of toxic,explosive, radioactive, or expensive sensitive materials on syntheses isreduced.

SUMMARY OF THE INVENTION

In accordance with the present invention, there are provided chemicalreaction apparatuses. These apparatuses comprise a reaction supporthaving a first surface (alternatively called a reaction surface)thereupon. The reaction surface is considered to have a plurality ofpreselected reaction sites upon it. These are generally in a regulargeometric array such as a grid, but may be in other patterns as well.The apparatus further comprises a first droplet generator for jettingreactant droplets upon the first surface. The apparatus furthercomprises a second droplet generator for jetting droplets of a secondreactant species upon the first surface. The droplets of each of thefirst and second droplet generators are under control of a control meanssuch as a general or special purpose digital computer, with attendantswitches and actuators, for causing the droplets from each of thedroplet generators to impact upon definable sets of the preselectedreaction sites on the reaction surface of the reaction support. Theimpacting of the droplets upon the sets of reaction sites is preferablyin accordance with a preselected pattern or patterns. In this way,chemical reactants can be directed to particular reaction sites upon thereaction surface in any desired order so as to achieve desired chemicalreactions at such reaction site without the need for removing,manipulating, and redeploying the reaction support.

In accordance with preferred embodiments, three, four, or more dropletgenerators are provided, each connected to one or more chemical reactantreservoirs, in order to provide diversity among the chemical reactionspossible at the reaction surface. The control means is preferablyprogrammed to deliver different sequences of reactants at differentreaction sites so as to synthesize differing chemical compounds at suchsites. Preferably, the methods of the invention are performediteratively such that relative complex molecules, such as oligomers, canbe synthesized.

Synthesis has been found to be benefitted by employment of porousreaction supports, especially those having the capability oftransporting fluid from the first (or reaction) surface to a secondsurface thereof. Improvement of yield through exploitation of theinternal pore volume of the support and amenability for improved productharvest is also now possible.

Once the different chemical moieties have been synthesized at theplurality of reaction sites, they may be employed in a variety of highlyuseful ways. Thus, the different chemical moieties may be exposed totest solutions, such as bodily fluids of a test animal, to diagnose orascertain bodily states in such animal. A wide variety of assays maythus be formulated using the apparatus and methodologies of the presentinvention. In accordance with other utilities, the chemical species thusformed may be used as probes in biological systems or as primers orsubstrates for polymerase chain reaction amplification or the like.Hybridization studies may also be conducted using apparatus and methodsin accordance with the present invention, where the products of themethodologies are oligonucleotides, polypeptides or other hybridizablespecies. All of the foregoing utilities are known per se to thoseskilled in the art.

In accordance with other preferred embodiments of the invention,reaction apparatuses are provided where one or more droplet generators,each in fluid communication with pluralities of reactant reservoirs, areemployed to perform synthesis. Control means are caused to effect theoperation of valving moieties to select reactants in appropriate ordersto achieve the desired reactions at individual reaction sites.

In accordance with the invention, methods for synthesizing chemicalspecies comprise identifying a plurality of reaction sites upon areaction surface and jetting upon a first set of such reaction sites,droplets of fluid comprising a first chemical reactant. The methods alsocomprise jetting upon a second set of such reaction sites, droplets offluid comprising a second chemical reactant species. It will beappreciated that, through practice of the present invention, the firstjetted reactant species and the second jetted reactant species may bejetted to the same or different reaction sites on the reaction surface.Control means for effectuating the jetting of such fluids upon theselected sets of reaction sites is also invoked to attain the ends ofthe invention. Iterative jetting of reactant species permits theelaboration of wide varieties of chemical moieties as the reaction sitesand permits the generation of libraries of diverse species.

It is also possible to employ the present invention in a hybrid fashionby combining it with other chemical reaction schemes. Thus, for example,a support for reaction may be coated with an initial reactant speciesand then reacted through the jetting of chemical reactants inpreselected locations on the surface. Subsequent reaction at theselected locations on the surface in accordance with the invention mayensue whereby a plurality of reactant species are delivered to suchlocations. Thus, by pretreating the support where the reaction is tooccur with a first reactant moiety, certain economies of scale may beattained. The employment of chemical jetting technology to deliver oftenexpensive reagents for at least some of the subsequent steps, is,however, preferred.

At any reaction site, either or both of the first and second reactantsmay be delivered thereto. Thus, at the reaction site, either the firstreactant, the second reactant, or the first reactant followed by thesecond reactant can be so delivered. The ensuing chemical reactions willdepend upon the identity and order of chemical reactants delivered toany particular reactant site.

This is also true where an initial reaction species is applied to thesupport prior to the delivery of jetted reactants. In such case, atreaction site R_(n), one of four situations will prevail after jettingof two reactants has occurred. First, it may be that neither the firstnor the second reactant are directed to a given reaction site. In thiscase, only the initial reaction species will have been delivered to thissite. In two additional cases, either the first reactant or the secondreactant, but not both is jetted to site. In this case, two differentcombinations of two reagents are delivered to the site. Finally, it maybe seen that both the first and the second reactant can be jetted to thesite whereupon three reaction species will have been so delivered.Persons of ordinary skill in the art will readily appreciate how acomplex series of reactants can be delivered to particular sites on areaction surface to achieve complex and varied chemical reactions. Thepresent invention is suited to the delivery of a large variety ofchemical species including reagents, intermediates, blocking anddeblocking agents, monomers, dimers, oligomers, solvents, washingagents, cleaving agents and the like.

In accordance with preferred embodiments, the methodologies of thepresent invention are performed iteratively. Thus, three, four, five,and more reactants can be delivered to a reaction surface in varyingcombinations at different reaction sites on the surface. The number ofdifferent chemical moieties which may, thus, be elaborated isextraordinarily numerous and varied. It is, thus, possible to generate,isolate and recover a wide variety of different chemical species in ahighly automated fashion on small reaction surfaces. The presentinvention also provides reaction assemblies wherein a reaction supportsurmounts a collection plate, preferably one having a plurality ofcollection wells. Transport of chemical species through the reactionsupport enables their collection in the collection wells where they maybe easily recovered, analyzed, tested, hybridized, screened, assayed andotherwise utilized. The efficient deposition of product species intosuch collection vessels is a particularly advantageous aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 are generalized drawings of droplet generating apparatususeful in the invention depicting salient features.

FIG. 3 depicts a single head droplet generating apparatus, in schematic,wherein a plurality of reagent reservoirs is in fluid communication withthe droplet generating head. Delivery of chemical species to a reactionsurface having a plurality of reaction sites is depicted.

FIG. 4 is a chemical jetting apparatus in schematic, wherein a pluralityof chemical droplet generating heads is employed to direct differentchemical reagents to sites on a reaction surface.

FIG. 5 shows the transitting of a chemical droplet generator over areaction surface to deposit droplets of reagent at preselected sites onthe surface.

FIG. 6 shows a reaction support surmounting a collection plate andcollection wells in the collection plate. The transport of liquidspecies from the reaction surface to a second surface of the reactionsupport and its collection into a collection well is shown.

FIG. 7A depicts one embodiment of the invention where reaction wells areformed in a shaped body for holding a reaction support. The wells funnelliquid species transitting the reaction support such that the same maybe collected by collection wells of collection plate.

FIG. 7B is a plan view of a portion of a shaped body containing reactionwells and reaction support, the whole suited for surmounting acollection plate.

FIG. 8 is a depiction of a preferred apparatus having a transittingdroplet generating head together with preferred reactive supports withinreaction wells of a shaped body. Collection wells in a collection plateare shown.

FIG. 8A shows reaction wells in a shaped body while FIG. 8B shows areaction support within a well.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides significant improvements in chemicalsynthesis and recovery technology. The apparatuses and methods of thepresent invention are advantageously employed in the preparation ofoligomers, especially oligonucleotides and polypeptides, and in thepreparation of libraries of compositions having diverse chemicalstructure. They are also useful for the synthesis of a wide variety ofnon-oligomeric molecules, especially those requiring hazardous orexpensive materials. In accordance with the present invention, it hasnow been found to be highly desirable to effect chemical reactions upona reaction support through the sequential jetting of chemical reagentspecies upon predefined sets of reaction sites on such surfaces. Theproblems associated with mounting and dismounting of reaction surfacesfrom the chemical jetting apparatus is avoided or minimized throughemployment of embodiments of the present invention.

FIG. 1 is a depiction of a chemical jetting apparatus which may be usedwith embodiments of the present invention. It will be appreciated thatchemical jetting apparatus suitable for use in the present invention maybe viewed as being essentially similar to apparatus used in "ink jet"printing. Ink jet printers are known per se and have achieved a separatestatus in the patent and other literature. For example, class 346 of thepatent classification of the United States Patent and Trademark Officecontains a large number of patents directed to ink jet technology, tomethodologies for employment of ink jets, and to apparatus for usetherein. All may be useful in the practice of this invention. Whilecertain modifications of basic ink jets are preferred for use inaccordance with the present invention--chiefly to render the same inertwith respect to the chemical reactants employed--the basic mechanicaland materials considerations which attend the provision of ink jetapparatus apply to the manufacture of chemical jetting apparatus aswell.

Referring now to FIG. 1, chemical jetting devices are conventionallyactuated through piezoelectric devices. A source of chemical reagent, 10is provided, conventionally through a pumping means, 12 to a chamber, 14in mechanical communication with a piezoelectric material, 16. Thechamber 14, is provided with one or more orifices, 18 through whichdroplets of reagent, 28 may be expressed through the controlled pumpingaction of the piezoelectric material. The piezoelectric device iscontrolled by a driver, 26 which, in turn, is controlled by controller,32. In some apparatuses, droplets, 28 are provided with an electriccharge in a chamber, 24 upon their emergence from the orifice and areaccelerated in one or more planes in an acceleration chamber, 26 underthe influence of an applied voltage controlled by the controller, 32. Itwill be appreciated that the overall effect of the foregoing arrangementis to provide a series of droplets at spaced intervals traveling inpredetermined vectors, as showing established by the controller inresponse to operator programming. It is well known to direct individualdroplets of liquid, 28 to various selected locations on a reactionsurface, 40 which, in embodiments of the present invention, is areaction surface whereupon chemical reactions take place. Droplets,which are not to be directed to particular locations on the surface are,in accordance with this embodiment, directed to a "gutter", 42 forenvironmentally approved disposal or recycling.

The foregoing method of providing droplets and directing the same toparticular locations on a reaction surface, with excess droplets beingdiverted to a gutter is known as a "continuous jet" type of device. Itwill be appreciated that the elements of reagent supply, pump,piezoelectric device chambers, orifices, electrodes, and the like--inshort, those elements which are required to produce droplets of chemicalreagent and to direct them in preselected directions--are conventionallyand are conveniently denominated a "jetting head" or, preferably, a"droplet generator." These two terms are used interchangeably in thepresent application. While droplet generators may not conventionally beconsidered to include chemical reservoirs, plumbing, controllers,connectors and the like, it will be understood that all such apparatusas may be required to effectuate the delivery of reagent droplets inaccordance with the present invention are included as needed and willnot necessarily be separately recited hereinafter.

Another form of chemical jetting is conventionally denominated a"droplet on demand" device. Such a device is depicted in FIG. 2. It willbe appreciated that rather than have a uniform stream of chemicalreagent droplets provided by the apparatus, unneeded droplets beingdirected to a gutter, in droplet on demand systems, droplets areprovided only when actually required for distribution to the reactionsurface, 40. While it is convenient to use electrostatic directing meansunder control of a controller to direct these drops, it is alsopossible, and in many cases preferred, to physically move the source ofthe droplets--the droplet generator--with respect to the reactionsurface or, vice versa, the reaction surface with respect to the sourceof the droplets, thus to deliver droplets to particular locations on thereaction surface in an imagewise, preselected fashion. Indeed,combinations of both directing techniques may also be employed. In someembodiments, it is possible to rely upon the impetus provided by thepiezoelectric material, 16 upon the reagent, forcing the same throughthe orifice with sufficient kinetic energy to impact the reactionsurface without additional acceleration. In any event, the piezoelectricdriver, 22 and whatever motion of the droplet generator or reactionsurface 40, may be required is under control of a controller, 32.

Irrespective of which form of chemical reagent droplet generator isselected, it will be appreciated that droplets of chemical reagent willbe provided at a reaction surface in a fashion which is preselected asto location. Thus, the reagents may be applied in an imagewise fashionto specific sites on the reaction surface. In accordance with certainpreferred embodiments of this invention, reagents are applied at sets ofsuch sites such as in an array defined upon the reaction surface. Thus,a matrix of sites is conveniently defined upon the reaction surface andone or more reagents jetted to sets or subsets of such sites in amountsand in orders of deposition consistent with the chemical synthesisdesired at each site. Any of the chemical jetting apparatus describedabove may be used for jetting the reagents to these sites and, indeed,any of the apparatus, methods, and materials which have been knownheretofore for use in conjunction with ink jet printing, which arecapable of jetting liquids to predefined locations on a surface, mayalso be used or easily modified for use in accordance with the presentinvention to deposit reagents at preselected sites upon a reactionsurface. It will thus be appreciated that the present invention is notlimited to any particular droplet generator or control means so long asthe droplet generator and associated control means are effective fordelivering the reagent to preselected locations on the reaction surface.

It is preferred to provide the chemical jetting head in materials thatare inert with respect to the chemical reagents being delivered. Thus,it is preferred that the apparatus be constructed of materials such asglass, ceramic, porcelain, inert plastic, inert or passivated metal, andother material which is consisted with the reagent to be jetted by theparticular jetting head and associated equipment. Persons of ordinaryskill in the art will have no difficulty in determining appropriatematerials for the construction of apparatus useful for the practice ofthe present invention upon consideration of the chemical and/orcorrosive nature of the chemical reagents to be dispensed by theapparatus. It is convenient to employ polytetrafluoroethylene (PTFE) andother relatively inert polymers for the storage, transmission, andjetting of chemical reagents in accordance with this invention.Stainless steel is another preferred material, especially for thejetting head itself, while glass finds great utility for the storage ofchemical reagents.

It will be appreciated that many piezoelectric materials are relativelychemically inert. Accordingly, selection of an appropriate piezoelectricmaterial for inclusion in the chemical jetting apparatuses of thisinvention will be a matter of routine as well. Polyvinylidine fluoride(PVDF) is one piezoelectric material which is relatively inert to mostchemical species and may be used in the practice of this invention.Certain other piezoelectric materials are metallic and are inert withrespect to many reagent and may, accordingly, also be used. All suchmaterials are contemplated hereby.

Prior attempts to employ droplet delivery for synthesis of chemicalspecies at a reaction surface have employed single chemical deliveryheads delivering a single reagent adapted for the deprotection ofchemical moieties found on the reaction surface. It will be appreciatedthat this is a highly inefficient technique and one in which theattainment of high density of chemical synthesis on the reaction surfacecannot easily be achieved. This is so, inter alia, because the reactionsurface must generally be submitted to other chemical treatments withreagents other than those provided by the droplet delivery device. It isgenerally necessary to dismount the reaction surface from the dropletdelivery apparatus for these further chemical treatments. Realignment isdifficult and time consuming, thus efficiency is lost.

FIG. 3 is directed to apparatus which provides great efficacy in thesynthesis of chemical species on reaction surfaces. A droplet generatinghead, 50, which may be of the continuous droplet, droplet on demand, orother form, is provided in fluid communication with a plurality ofchemical reagent reservoirs, 52. Valving means, 54 is also provided andthis means is preferably electromechanical actuatable under control of acontroller, 56. In accordance with preferred embodiments, the chemicaldroplet generating head, 50 is traversable in one or more directionsthrough electromechanical means as is known for use in connection withink jet printing heads. Such traversing means, 58, which is optional butpreferred, permits the deposition of droplets of reagent at reactionssites, 42 on reaction surface, 40. Traversing through x or xy planes maybe performed as convenient.

As will be appreciated, use of the present invention will permit theautomation of chemical syntheses since the delivery of reagents in asequential fashion to any particular reaction site on a reaction surfacecan be controlled through controllers such as general purpose digitalcomputers or special purpose digital computers or processors. While itis not essential that the steps of the reaction sequence be controlledelectromechanically by control means, this is generally preferred. Thus,while the valving means traversing means, etc, 54 can be actuatedmanually and still fall within the spirit of the present invention, itis greatly preferred that control means e.g. a computer, actuate thevalves electromechanically and traverse the droplet generator inaccordance with programmed demands for particular reagents. A widevariety of reagent storage, valving, plumbing and other ancillaryapparatus may be employed within the spirit of this invention so long asthe same are generally inert with respect to the reagents in contactwith them. Similarly, the apparatus may be encased in a specialatmosphere, may be operated with exclusion of light or moisture, and maybe oriented in any convenient direction as may be preferred for anyparticular synthesis. All such modifications are contemplated hereby.

It will be appreciated that it may be necessary to rinse or otherwisepurge the chemical jet reaction system when different chemical reagentsare selected for use. Thus, it is preferred to provide appropriatesolvent means effective to remove one reagent from the system prior tothe provision of a second reagent thereto. Persons of ordinary skill inthe art will have no difficulty selecting appropriate solvent means andwashing steps to effect this goal. Unwanted materials may be jetted to adump or gutter for recycling or disposal.

As shown in FIG. 4, it is possible to avoid the need for all or some ofthe washing/purging steps which are generally required when pluralitiesof reagents are jetted from a single head. This may be accomplished inaccordance with a further embodiment of the present invention throughthe employment of a plurality of droplet generators which are eachcapable of delivering droplets of reagent to reaction sites, 42 on areaction surface, 40. Thus, a plurality of droplet generators, 50 areprovided in fluid communication with reservoirs of chemical reagents, 52through the mediation of valving means, 54. It is preferred that thedroplet generators, 50 and valving means, 54 be under control of acontroller, 56 which is preferably either a general purpose digitalcomputer, special purpose digital computer, or specialized controller.

The droplet generators, 50 may preferably be arranged in such a fashionthat they can traverse through a space while distributing reagentdroplets, however this is not obligatory. It is convenient to employfour jetting heads although other pluralities may be selected. Four ispreferred since four head ink jetting systems for color printingpurposes are known as are control means therefore.

In accordance with certain preferred embodiments of this invention,composite droplet generators may be employed. A composite dropletgenerator is one which incorporates a plurality of orifices within onephysical structure. Thus, the elements needed to effect a jetting ofreagent droplets may be integrated in such a fashion that a plurality ofdroplet orifices share one or more elements such as reagent reservoirs,pumps, piezoelectric elements and the like. All such modifications arewithin the spirit of the present invention.

FIG. 5 depicts a preferred aspect of the droplet generator relationshipto the reaction support. One or more droplet generators, 50 may becaused to be traversable with respect to the surface of the reactionsupport, 40. Such droplet generators may traverse along one axis or,preferably along two axes in the nature of a plotting device. In such acase, it is not generally necessary to employ electrostatic direction ofdroplets to reaction sites, rather the transverse movement of thedroplet generator can precisely place reagent droplets as required atparticular sites, 42. It is preferred that the droplet generators beoriented to direct droplets in a downward direction; the reactionsupport is preferably oriented horizontally. This permits gravity toassist in the penetration of reagent droplets into the pore volume ofpreferred reaction surfaces and to aid in transport of liquid throughsuch supports as will be described more completely infra in connectionwith other preferred embodiments.

In accordance with other embodiments of the present invention, reactionsupports are provided which are especially suitable to the practice ofthe methods of this invention. It will be appreciated that thetechniques as described in the present invention serve to deliverdroplets of chemical reagents to localized reaction sites on thereaction surface. It has now been found, however, that great advantagesmay attained by employment of particular reaction substrates havingsignificant internal surface area as reflected by pore volume.Accordingly, it is one aspect of the present invention to employreaction supports for chemical jetting systems, which reaction supportshave significant pore volumes. Such reaction supports are best describedby what they do. Their character of porosity leads to their ability tocause droplets of reagent to enter into the body of the substrate,within pores or voids, such that relative large amounts of fluid can beso accommodated. While objects of this invention can be accomplishedwith reaction supports having relatively little porosity, the largereffective surface areas available from relatively porous substrates isdesirable. By employing porous reaction supports in accordance with thepresent invention, it may be seen that increased volumes of reagent maybe delivered to reaction sites on reaction surfaces since the reagentliquid will be absorbed into the pore volume of the reaction supportthrough capillary action, gravity, and otherwise.

It is preferred to employ reaction supports having a porosity such thatliquid is capable of passing from the surface upon which reagentimpinges to the opposite surface, e.g. the support is at least partlypermeable. Examples of such permeable reaction supports includepermeable and semipermeable membranes, especially isotropic andanisotropic, polymeric membranes, and control pore glass. Poratedmembranes and glasses, e.g. having surface which have been subjected toion nuclear bombardment to effect holes therethrough, and variousceramics are also useful. Such materials are best described by what theydo rather than by what they are. Thus, preferred reaction supports arethose having a significant pore volume and which are not inconsistentwith reaction chemistries to be practiced upon them. Accordingly,employment of a reaction support which reacts with or destroys any ofthe chemical species which are intended for contact with it would becontraindicated for that particular chemical reaction sequence. It isbelieved that persons of ordinary skill in the art will have nodifficulty in determining appropriate reaction supports in accordancewith the foregoing principles.

Exemplary reaction supports for use in accordance with the presentinvention include CPG (controlled pore glass) available from variousdistributors including CPG Inc./Millipore Corp.; RAPP copolymer, ahighly crosslinked polystrene, sold as TentaGel or a like product HLP(high loaded polystrene) sold by ABI Corp.; Primer Support, a highlycrosslinked polystrene, sold by Pharmacia; POROS-OS polystrene sold byPerSeptive, MPG (a magnetic pore glass) sold by CPG Inc.; Nucleic AcidMembrane Support sold by Millipore. Other useful supports includemembranes sold by the Amicon division of W. R. Grace, Inc., and thosesold by Gelman Sciences. Polyether sulfone, polysulfone, PVDF, PTFE,PVC, polypropylene and nylon supports may be employed for variousapplications, as may many other materials.

Other membrane supports include membranes as described or referenced inU.S. Pat. No. 4,923,901 assigned to Millipore Corp.; various supports asdescribed in patent application WO 94/05394 and references citedtherein; and various supports as described in patent application WO90/02749 including activated polystrene layer on a polyethylenemembrane. Further Zeolites, cellulose, cottons, other polystyrenes thatcan optionally be crosslinked, polyacrylamide, which may be optionallycrosslinked, latexes, dimethylacrylamide optionally crosslinked withN,N'-bis-acryloylethylenediamine can also be mentioned.

For certain preferred embodiments of the invention, it is preferred thatthe reaction support possess porosity in such a fashion thatindiscriminate blotting or wicking is avoided. Indiscriminate blottingor wicking in this context is defined to mean the wicking away of liquidfrom the point of impingement of a chemical reagent upon a reactionsurface in a direction in other than the direction normal to and throughthe surface. Thus, it is desired that the reaction supports transmitliquid impinging upon their surface through the body of the support andto the opposite side rather than across the surface or laterally intothe body of the support. While, inevitably, some wicking of reagent willoccur, it is preferred that the fluid be transmitted predominantly inthe normal direction. This concept of wicking is necessarilyqualitative, but is believed to be understood by persons of ordinaryskill in the art.

Supports which exhibit a diminished tendency towards laterally wickingor blotting and which transmit fluid normal to the surface and throughthe support, are greatly desired for a number of reasons. First,reliable washing of the support upon which reagent is absorbed can beattained more easily when lateral wicking is avoided. Moreover, whenlateral wicking is minimized, reaction sites can be defined more closelytogether on a surface of a reaction support than if lateral wicking issignificant. The selection of reaction supports for use in conjunctionwith the present invention having diminished tendencies towards lateralwicking while maintaining high internal pore volume permits greaterefficiencies in performance of the methods of the present invention.Thus, greater synthetic speed and yield may be evidenced since it iseasier to wash the surfaces contacted by the chemical reagents whilemaintaining a high concentration of reaction sites for reaction.

Capillary glass has been found to be particularly useful for thepractice of the present invention. Capillary glass is a material whichis known per se and comprises substantially parallel glass filamentsoriented in a direction substantially normal to the surface. Variouscapillary glasses are commercially available such as those that are usedfor capillary gel electrophoresis. These include both internally coatedand uncoated supports available in various internal diameters including50, 75 and 100μ that typically have a 365μ outside diameter. One suchsupport is available for Polymicrotechnologies, Inc. Such materials haveeffective large internal porosities compared to surface area and havelittle tendency toward lateral wicking. "Bundles" of such capillaryglass filaments are be assembled for use as support of the processes ofthe invention.

A wide variety of other materials may also be used in accordance withthe present invention. The 48 well and the 96 well versions ofGibcoBRL's "The Convertible Filtration Manifold System" sold by LifeTechnologies, Gaithersburg, Md. can be used as separators for reactionarea on an appropriate planar membrane such as the above mentionedNucleic Acid Membrane Support sold by Millipore, Corp. In essence, thefiltration manifold defines reaction areas that can be filtered undervacuum to facilitate reagent and solvent removal. The jetting heads ofthe invention can be used to create a single polymeric species withineach of the areas defined by the top plates of these filtration devicesor they can be used to create multiple polymeric species within eacharea. Thus in one embodiment of the invention, such a manifold, as forinstance the 96 well manifold, will be used in conjunction with thejetting head of the invention to create 96 individual polymeric specieswhereas in a further embodiment, the jetting head will deposit, as forinstance, as 10 by 10 matrix of individual polymeric species in eachwell of the manifold to create a total of 9,600 individual polymericspecies over the 96 wells of the manifold.

Further preferred support structures of the invention utilize an arrayof openings in a matrix support material. Such structures can be formedutilizing Helix HD-864-PS-50 or HD-864-PC-50 864 Well High DensityMicrowell plates (having 864 individual wells, each of a 20 μl volume,located within the foot print size of a normal 96 well microtiter plateavailable from Helix, San Diego, Calif.) loaded with an appropriatereaction support medium. Each well so modified serves as an individualreaction vessel that can be charged via the jetting device withappropriate reagents, wash solvents and the like.

These same Helix high density microwell plates can be modified byremoving the totality of the bottom surface of the plates by machining.This creates a structure having a plurality of parallel capillary tubessuitable of loading with an appropriate reaction support medium. Suchmodified plates can be used with an appropriate commercial vacuumapparatus such as the above described GibcoBRL Filtration ManifoldSystem from Life Technologies, Gaithersburg, Md. The modified highdensity microtiter plates are supported on a gasket having a holepattern that matches the hold pattern of the modified plate. In oneembodiment of the invention, strips of the above-noted Nucleic AcidMembrane Support sold by Millipore are rolled, in a manner like a cigar,and are inserted in the so formed capillary tubes. Upon completion ofsynthesis of the polymeric compounds, the strips can be remove with thepolymeric materials still attached and used as such for biologicaltesting. In a further embodiment, the polymeric materials are releasedfrom the support membranes and used in solution. Alternately such highdensity microtiter plates can be modified by the drilling an opening, asfor instance a 0.1 to 0.5 mm opening, in the bottom of the individualwells. A porous plug is then located in the bottom of each well and thewell loaded with an appropriate support medium.

In a further preferred support, glass whiskers can be repeated sonicatedto create porosity therein. Such porous whiskers are then alignedaxially and are imbedded in a spaced array in an inert matrix material,as for instance polyethylene, to form a filamentous structure having aplurality of parallel aligned porous glass rods. These porous glass rodscan then be derivitized with linkers. Usable as a linker is one of themany linkers known for use with CPG and other glass supports. Thelinkers, in turn, are used to attach the first monomer unit of aoligomeric compound in the same manner as is practice with common CPGsupport materials.

Further preferred support materials are anisotropic polymeric membranes.These are known per se and are widely available such as from the AmiconDivision of W. R. Grace. Such anisotropic membranes have a first surfacewith relatively "tight" pores which communicate with increasingly largerpores in the membrane. At the distal surface of the membrane, the poresare quite large and provide no hydraulic impedance of fluid movement.Application of chemical reagent to the tight surface of such membranes,called the skin, with concomitant migration to the large pore volumesjust below the surface is highly advantageous in the practice of thisinvention. Such membranes are available in a large number of chemicalforms including polyether sulfones, polysulfones, polyvinylidenefluorides, nylons, PTFEs, acrylics and many others. Such materialsgenerally exhibit a desirably small lateral wicking and are,accordingly, preferred for use in some embodiments of the invention.

Other preferred membrane supports utilizes a polyvinylidene difluoridemembrane as described above that is a treated with diaminopropane in DMFe.g. utilizing the procedures of Example 1 of U.S. Pat. No. 4,923,901 toform a polymeric membrane suitable for use to form polynucleotide typeoligomers. A further particular preferred membrane, particularly usefulfor synthesis of peptide and peptide like (peptoid, polycarbamate andthe like) polymeric compound libraries, utilizes a polypropylenemembrane that is derivitized with hydroxypropylacrylate by coating thepolypropylene membrane with crosslinked polyhydroxylproplyacrylate. Thismembrane was described by Daniels, et. al, in poster # T81 at theProtein Society meeting, San Diego, Calif., 1989. Particularly usefulfor synthesis of oligonucleotides and the like is a membrane supportdescribed by Fitzpatrick, et. al., presented in a paper entitled"Membrane Supports for DNA Synthesis", at the 1993 "Innovations andPerspectives in Solid-Phase Synthesis" conference at the University ofOxford. This support utilizes a PTFE (polytetrafluoroethylene) membranethat is coated with a terpolymer coating consisting ofmethylene-bis-acrylamide, N,N-dimethylacrylamide andaminopropylmethacrylamide. Nitrophenylsuccinates of appropriate firstmonomeric units are reacted with the primary amine groups of thecoating. The density of the growing oligomer is controlled by thespacing of the aminopropylmethacrylamide monomer of the coating. Sterichinderance can be prevented by infrequently incorporating this monomerin the coating.

It will be appreciated that syntheses similar in many chemical respectsto existing "solid state" synthesis are preferred for use in connectionwith some embodiments of this invention. In such cases attachment of aninitial reactant, chemical substrate or moiety to a solid, here thereaction surface of the reaction support, is preferred. The materialsselected for the reaction support should preferably be capable offunctionalization by such an initial chemical moiety. A large variety ofappropriate materials are known in this context such as glasses,ceramics, many polymers and other species. Following synthesis, it isgenerally the case that the synthesized chemical species areconventionally cleaved from the solid and recovered through washing.Selection of reaction supports stable to such practices is greatlypreferred.

It is also preferred in some embodiments to effect partial synthesis ofchemical species. Thus, for example, oligomers can be elaborated throughiterative solid phase chain elongation reactions and then cleaved andrecovered. Post processing, such as to remove protecting groups, maythen be performed as desired.

A matrix of reaction sites is preferably defined on the surface of thereaction support such sites being intended for the deposition ofdroplets of chemical reagent. Such sites are not generally markedvisibly, but rather are defined geometrically and addressably by thecontrol means for deposition of reagents. Pluralities of sites may beused for the same reaction series or different reactions may occur ateach in accordance with the designs of the operator.

In accordance with other embodiments of the invention, chemical reactionapparatuses are provided that are particularly adapted to the presentmethods. A reaction support having a reaction surface for the desiredchemical reactions is provided. A collection plate is also provided,such plate being adapted for lying adjacent to the reaction support atthe surface distal from the reaction surface. The collection plate isalso preferably provided in such a fashion as to have a plurality ofcollection wells which are arrayed in a matrix isomorphic with thematrix of reaction sites extant upon the reaction surface of thereaction support. The assembly is such that when a fluid is placed uponthe reaction surface of the reaction support at a reaction site thereof,it will pass through the reaction support, arrive at the second surfacedistal from the first surface, and collect in a well of the collectionplate corresponding to the reaction site. It is apparent that the arrayof reaction sites and collection wells on the collection plate may bestructured in any geometric pattern as may be convenient. A rectangulararray is convenient and a conventional ninety-six well plate can be usedto good effect.

It is also possible and, in some cases preferred, to associate reactionsites with collection wells in a fashion other than one-to-one. Forexample, a plurality of reaction sites may be identified which areassociated with a single collection well on the collection plate. Forexample, a matrix of reaction sites on the surface of the reactionsupport may lead to a common collection well. This may be done for anumber of reasons including the desire to improve the volume ofchemicals processed. A further, and in some cases preferred utility forsuch an arrangement is to provide libraries of chemical speciescollected within particular collection wells. Accordingly, a matrix ofreaction sites, e.g. a 10×10 matrix, can be associated with a singlecollection well and the reagents jetted to the 10×10 matrix controlledboth as to identity and timing in such a manner as to provide onehundred different chemical moieties to be collected in a singlecollection well associated with the 10×10 matrix. Since the sequence andidentity of chemical reagents jetted to the particular reaction sites isknown in all cases, the same having been determined through theprogramming of the control means, the composition of the chemicallibrary resident in the particular collection well is known withcertainty. It is thus possible, in the example given, to assay onehundred chemical species in a particular chemical, biological, or otherassay, knowing with certainty the identity of all one hundred chemicalspecies whose performance is to be monitored in the assay. Chemicallibraries are inherently useful and valuable. The same are in greatcommercial demand and should be viewed as a commercially useful articleper se.

The foregoing library procedure is of obvious benefit in the screeningof new drugs and diagnostics. It is also of great benefit in theidentification of chemicals which have agricultural, therapeutical,medicinal, industrial, or other practical uses. Indeed, the presentinvention provides an unambiguous, rapid, and powerful method for thegeneration of such libraries without the ambiguity that random synthesisprovides. As such, it represents a great advance over prior methods forlibrary creation.

FIG. 6 shows one example of a preferred arrangement. Reaction support,40 surmounts collection plate 44 having collection wells 46. Thereaction surface, 43 of reaction support, 40 is impinged by reagentdroplets, 20 at one predefined reaction site, 42 on the reactionsurface, 43 of the reaction support, 40. Internal porosity, 47 is shownalthough the exact geometry of such pores will rarely be known. Suchpores or voids may be those from nuclear bombardment from anisotropicsynthesis, or as otherwise known or as described herein. In any event,such pores preferably communicate with the second, distal surface, 45 ofthe reaction support, 40 preferably without undue lateral wicking, suchthat liquid impinging a particular site, 42 on the reaction surface, 43of the reaction support will be transported to the collection well, 46of collection plate, 44 which is isomorphic with such reaction site.Such liquid is indicated, 49.

FIGS. 7A and 7B are cross section and plan views of additional preferredembodiments of this invention. In this embodiment, the reaction supportis present in subportions located within reaction wells, 62 on a shapedbody, 60. The shaped body is any solid inert with respect to thechemical reactions to take place and capable of appropriate shaping,sterilizing, cleaning and the like as may be desired. Polymer, e.g.nylon or PTFE, glass, ceramic or metal are exemplary materials. Thereaction wells, 62 are preferably molded or machined into a surface ofthe shaped body, 60 in any convenient manner, such as by milling. Thereaction wells are preferably in fluid communication with a secondsurface of the shaped body, and are optionally but preferably adapted tofunnel liquid from a larger portion of the well to a smaller or funnelportion, 64. The funnel portions are preferably located to cooperatewith collection wells, 46 of a collection plate 44 adapted to lieadjacent the shaped body distal from the reaction wells. Chemicalreactions are performed on the reaction support, 40 in each well whichcan be easily rinsed through the well funnel portions. Followingcompletion of synthesis, the completed chemical species can be directedthrough the funnel portions for collection in the collection wells.While the apparatus thus described is useful with chemical jettingtechniques as set forth herein, other synthetic techniques may also beused therewith.

FIG. 7B shows a plan view of a portion of a shaped body, 60 with onecomplete well, 62. An array of reaction sites, 42 are shown. Thereaction support may be any material herein described or, indeed, anyother as may be benefitted from the reaction well and funnel aspects ofthe invention.

FIG. 8 is a schematic depiction of one system in accordance with thisinvention. Chemical droplet generating head, 50, in this depictionattached to a plurality of reagent sources, 52, is arranged to depositchemical species upon reaction surface, 43, of reaction supports, 40disposed in reaction wells in first surface, 63 of shaped body, 60. Inaccordance with this embodiment, pluralities of matrices of reactionsites, here 10×10 sites, shown in FIG. 8A, are defined on the reactionsurface of the reaction supports, 40 such that each set of one hundredreaction sites generally communicates with one area on the second,distal surface, 65 of shaped body 60. A collection plate, 44 havingcollection wells, 46 is also provided in an isomorphic fashion suchthat, in this example, for each 10×10 matrix of reaction sites leadingto a generally common area on the distal surface, 65 of the shaped body,60, one well is provided into which liquid from the reaction sites canflow in a common fashion. FIG. 8B depicts reaction support, 40 in a wellof shaped body, 60.

Throughput of liquid from the reaction sites to the second surface ofthe reaction support may optionally be encouraged in a number of ways.In accordance with preferred embodiments, a partial vacuum is applied insuch a fashion that liquid is extracted from the reaction support.Subsequent washing with an appropriate solvent can ensure completetransfer of material from the reaction support to the collection plate.Alternatively, application of solvent to the reaction sites on thereaction surface of the reaction support will have a washing effectcausing translocation of chemical species to the collection wells.

Once the products of the chemical reaction have been transferred to thecollection wells, they may be used in further synthesis, may be used perse, may be subjected to one or more assays, or may otherwise be employedin many ways known to persons of ordinary skill in the art.

In accordance with certain preferred embodiments, it is preferred toadapt the apparatus of the present invention so as to direct liquid fromthe reaction support to the collection wells in as highly efficient afashion as possible. Thus, the reaction support may be formed of acomposite or may be adapted in various ways to encourage this goal. Itis within the spirit of the present invention to provide compositesupports for elaboration of chemical species. Thus, a shaped bodycontaining pluralities of wells may be elaborated, such wells beingshaped so as to effectively funnel liquid from a first surface to asecond surface thereof as described above. This arrangement is adaptedeither to be surmounted by a reaction support where chemical reactionsare to take place or to contain in reaction wells, reaction support forsuch reactions. In any event, the elaboration of appropriately shapedstructures facilitates the cooperation of reaction support withcollection wells in a collection plate to accomplish preferred goals ofthe present invention. It will be appreciated that large numbers ofvariations are possible in this context and all are within the spirit ofthis invention.

It will be appreciated that these aspects of the invention are notlimited by the particular type of chemical synthesis that may beemployed and while both single head jetting systems as well as pluralhead jetting systems are contemplated hereby other reagent applicationtechniques are also comprehended by this invention.

It will also be understood that a wide variety of atmospheres may beimposed upon the synthetic methodologies of the present invention topermit the elaboration of chemically sensitive, corrosive or reactivemoieties. Thus, it is often preferred to employ an inert atmosphere,such as argon, and to exclude moisture.

Determination of preferred conditions of time and temperature as well asreagent concentration and catalysis is within the skill of the routineerin the synthetic art. For the synthesis of oligomers, conditionsgenerally similar to those employed in automated synthesis equipmentknown heretofore provides a useful point of departure from whichselection of appropriate conditions may easily be determined for anyparticular apparatus or method in accordance with this invention.

In accordance with some embodiments it is preferred to orient thesynthetic systems such that the effects of gravity may be invoked totransmit liquid from the reaction sites through the reaction support tothe second surface of the reaction support for collection in collectionwells. It will also be appreciated that evaporation of solvent from thecollection wells may be accomplished through the application of gas topermit sequential washing steps and the like to occur withoutoverflowing the collection wells.

Through the use of the collection well embodiments of the presentinvention, interface of the methodologies of this invention withpreviously known techniques in chemistry, biotechnology, and otherdisciplines may be attained. Thus, once predetermined species are knownto be present in collection wells of the collection plate, varioustraditional analytical techniques may be applied. These include testingprotocols based upon cell based and biochemical assays. These can beused in association with other techniques such as ELISA assays and thelike. Testing protocols can be used to measure various parametersincluding enzyme/substrate interaction, protein/protein interaction,substrate/transcription factor interaction, ligand/receptor interaction.

A very important use of the present invention is for the generation ofhighly accurate, libraries of chemical compounds, specially oligomericlibraries. Exemplary uses of such libraries are abundant. Illustrativecell based assays and brief descriptions of the assays are:

HIV--CEM-SS cells are infected with live virus (HIV-1) in presence oflibrary subsets; assay measures protection of the cells by the librarysubsets from virus-induced cytopathic effects. TuberculosisBacteriocidal effects of the library subsets on the mycobacterium aremeasured.

Tumor Necrosis Factor--Inhibition by library subsets of TNF induction ofinflammatory cascade in NHDF cells is monitored using ICAM-1 inductionas endpoint.

Interleukin 1-Beta--Inhibition by library of IL1-β induction ofinflammatory cascade in NHDF cells is monitored using ICAM-1 asendpoint.

LPS--Inhibition by library subsets of LPS induction of inflammatorycascade in NHDF cells is monitored using ICAM-1 induction as endpoint.

Malaria--Inhibition of parasite replication in blood cells by librarysubsets is measured.

Interleukin-6--Inhibition by the library subsets of interaction of IL-6and its receptor expressed on live cells is monitored using an antibodyspecific for IL-6.

MRP/MDR--Enhancement by the library subsets of the toxic effects ofchemotherapeutic drugs on mammalian cells expressing either MRP or MDRIis monitored using an MTT assay.

PDGF--Library subset inhibition of the radioactively-labeled ligandinteraction with membrane-bound receptor is measured. The membrane ispartially purified from guinea pig spleen. Complement C5_(A) Librarysubset inhibition of the radioactively-labeled ligand interaction withmembrane-bound receptor is measured. The membrane is partially purifiedfrom guinea pig spleen.

LTB₄ --Library subset inhibition of the radioactively-labeled ligandinteraction with membrane-bound receptor is measured. The membrane ispartially purified from guinea pig spleen.

PLA₂ --Library subset inhibition of enzymatic activity of type IIphospholipase A₂ is measure. The substrate is E. coli with aradioactively-labeled fatty acid in the membrane.

TAT/tar--Biotinylated TAR RNA is bound to streptavidin-coated wells of a96-well microtiter plate. Inhibition by the library subsets of theinteraction of the tat protein with the TAR RNA is monitored in anELISA-type assay using a tat-specific antibody.

In addition to the above specific uses, other use for the compounds thatcomprise the libraries of this invention are as general use enzymeinhibitors, additives for foodstuffs, as agent used in affinitychromatography, and as probes and diagnostic agents in kits and the likewith or without the addition of suitable labels including fluorescentagent, radioactive agents, or enzymes labels. Other uses will also beapparent.

As will be appreciated, the apparatuses and techniques of the presentinvention are amenable to a wide variety of chemical and biochemicalsynthetic schemes. Thus, it is possible to employ nearly any type ofchemical reaction save, possibly, those which take place exclusively inthe gaseous phase and those which require biphasic catalysis. Ingeneral, it is preferred to attach a first species to the reactionsurface, both at the actual surface of the reaction support and to agreater or lesser extent at the surface of the internal pore volume,followed by subsequent chemical reactions. It is convenient to attachthe first chemical reactant universally over the entire reaction supportsince the same may be accomplished through immersion of the support inappropriate reactants. Subsequent, dropwise application of reagentseffect chemical reactions locally at the reaction sites under thecontrol of control means. It is also possible and in many applicationspreferred, to perform all reactions in a dropwise fashion throughchemical jetting.

Oligomers are preferred species for synthesis in accordance with thepresent invention. Thus, oligonucleotides, polypeptides, andoligosaccharides may be so synthesized. Such oligomers may be eithersynthesized in relative bulk, where many or all of the reaction sitesare caused to experience the same reaction conditions and to lead to thesame reaction products, or may be individually determined for subsets ofreaction sites.

In accordance with other embodiments, it is preferred to employ theapparatuses and methods of the present invention to preparenonoligomeric chemical moieties. Thus, "classical" chemistry may beemployed to synthesize such chemicals either "in bulk" or with differentchemical species being synthesized at different subsets of reactionsites. While it is preferred in some cases to physically attach thedeveloping chemical moiety to the reaction surfaces during the course ofsynthesis, it is also possible in some embodiments to avoid this step.In such case, it is generally preferred to effect careful reaction andwashing conditions so as to retain the intermediate chemical species onthe surface for further reaction. This may generally be done throughcareful selection of the reaction support, chemical reagents, andwashing solvents in view of the molecules to be synthesized. Persons ofordinary skill in the art will know how to effect such selections inview of the objects to be attained in particular synthesis.

It is believed to be possible to effect asymmetric synthesis throughemployment of the present invention. If a chiral synthetic support isadopted, in some circumstances growing chemical intermediates will adoptpreferred stereochemical configurations leading to the synthesis of oneenantiomer over another without the use of chiral reagents. Theasymmetric synthesis of chemical species on asymmetric surfaces is knownper se and such techniques may be adopted here. The methods of theinvention may be used to produce oligomeric species having a variety ofdifferent monomeric subunits. Thus, the methods of the invention mayadvantageously be employed in the synthesis of polymers of nucleotides(oligonucleotides or nucleic acids), peptides, proteins, peptide nucleicacids (PNAs), and other polymeric species synthesizable by iterativeaddition of synthons to adducts on the reaction surface.

Synthetic techniques such as solid phase peptide synthesis and solidphase nucleic acid synthesis utilize the ability to selectively protectand deprotect specific functional groupings. Protecting groups areconveniently characterized as either "temporary" or "permanent.""Temporary" protecting groups are quantitatively removed at each step ofthe synthesis to allow coupling of the next synthon. "Permanent"protecting groups are stable to the conditions of the iterativeelongation cycle, and therefore protect side chain, nucleobase, or otherfunctional groups which do not participate in, but may interfere withchain elongation. Typically, permanent protecting groups are chosen suchthat conditions required for their removal are equivalent to thoserequired for cleavage of the completed chain from the reaction support,affording concomitant removal.

The methods of the present invention may be used to synthesize peptidesby standard solid phase peptide synthesis (SPPS) methodologies (see,e.g., Merrifield, J. Am. Chem. Soc., 1963, 85, 2149 and Science, 1986,232, 341). Media suitable for use as reaction supports in connectionwith peptide and synthetic applications of the invention includeaminomethyl polystyrene resins, various polyamide support materials,membranes, cotton and other carbohydrates, controlled-pore silica glass,and other media known to those in the art for use in peptide synthesisas solid supports. See Synthetic Peptides A Users Guide, Gregory A.Grant, Ed. Oxford University Press 1992.

The initial functionalization of the reaction support may be achieved byany of the more than fifty methods which have been described inconnection with traditional solid-phase peptide synthesis (see, e.g.,Barany and Merrifield in "The Peptides" Vol. 2, Academic Press, NewYork, 1979, pp. 1-284, and Stewart and Young, "Solid Phase PeptideSynthesis", 2nd Ed., Pierce Chemical Company, Illinois, 1984).

Typically, SPPS (solid phase peptide synthesis) is performed in the "Cto N" direction. Thus, anchoring linkers are designed such that cleavageat the end of the synthetic regime produces a C-terminal acid or amide.In preferred embodiments a linker containing an activated carboxyl groupis keyed to amino groups on the reaction support. In some preferredembodiments of the invention protected amino acid derivatives havinglinkers attached (so called "preformed handles") are keyed to thereaction support. See Synthetic Peptides A Users Guide, supra, at pages105-119.

Any of the several "temporary" protecting groups routinely used in theart are suitable for use in the present invention. Preferred among theseare the widely used BOC (t-butoxycarbonyl) and FMOC (N.sup.α-9-fluorenylmethyloxycarbonyl) groups. Other suitable amino protectinggroups include 2-(4-biphenyl)propyl 2!oxycarbonyl (Bpoc),2-(3,5,-dimethoxyphenyl) propyl 2!oxycarbonyl (Ddz),1-(1-adamantyl)-1-methylethoxycarbonyl (Adpoc) and4-methoxybenzyloxycarbonyl (Moz). Other suitable protecting groups willbe apparent to those skilled in the art, based on their experience andknowledge.

After keying of a linker and/or first monomeric synthon to the reactionsupport, the iterative process of chain elongation occurs. This mayproceed, in accordance with the invention, by any of the several methodsknown in the art for the formation of peptide bonds. Representative ofsuch methods are the use of in situ coupling reagents, active esters,preformed symmetrical anhydrides and acid halides.

Representative in situ coupling reagents suitable for use in the presentinvention include N,N'-dicyclohexylcarbodiimide (DCC), andN,N'-diisopropylcarbodiimide (DIPCDI), especially in conjunction withthe use of scavenging agents (so called accelerators or additives) suchas 1-hydroxybenzotriazole (HOBt), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU). A list of suitable in situ coupling agents may be found inSynthetic Peptides A Users Guide, supra.

Appropriately selected sites on the first surface of a reaction supportare jetted with a solvent as a washing or rinsing step, and then thetemporary protecting group on the terminal synthon is cleaved by jettinga deprotection reagent onto the preselected sites of the reactionsupport. This is optionally followed by the jetting of one or moreconventional rinsing reagents. The next protected monomeric synthon isthen jetted in a suitable solvent such as dimethyl formamide. Couplingor activating agents, including accelerators or additives such as HOBt,optionally may be jetted with the protected monomeric synthon oralternatively may be jetted independently in an appropriate solvent.

After a suitable reaction time, the iterative cycle is repeated untilthe desired amino acid sequence is achieved. Cleavage of the completedproduct from the reaction support is achieved by jetting a cleavingreagent onto the reaction support surface. The cleaving reagent alsotypically functions to remove the "permanent" protecting groups from theamino acid side chains. Choice of a specific cleavage reagent willnecessarily be determined by the particular synthetic chemistryemployed. For example, Boc SPPS chemistry will typically employ strongacid such as hydrogen fluoride for cleavage, while in Fmoc SPPSchemistry, the same result is typically accomplished withtrifluoroacetic acid. See Synthetic Peptides A Users Guide, supra, atpages 130-136.

The methods on the invention may similarly be employed in the synthesisof non-peptide polymeric species which consist of monomers linked bytraditional peptide chemistries. Representative of these species arepeptide nucleic acids (PNAs), which are disclosed in WO 92/20702. InPNAs ligands are linked to a polyamide backbone through aza nitrogenatoms. U.S. application Ser. No. 08/054,363, filed Apr. 26, 1993 and acorresponding PCT Application PCT/IB94/00142 filed Apr. 25, 1994discloses peptide nucleic acids in which their recognition moieties arelinked to the polyamide backbone additionally through amido and/orureido tethers. Additional PNAs and methods for their synthesis are alsodisclosed in U.S. application Ser. No. 08/088,658, filed Jul. 2, 1993and a corresponding PCT Application PCT/US94/07319 filed Jul. 2, 1994.

These PNAs are synthesized by adaptation of standard peptide synthesisprocedures. The synthons used are unique monomer amino acids or theiractivated derivatives, which are protected by standard protectinggroups. Thus, the synthesis of these PNAs may be accomplished accordingto the methods of the present invention in similar fashion to protocolsspecified above for synthesis of peptides.

For example, the reaction support may be functionalized according tomethodologies specified above to incorporateBoc-L-Lys(2-chlorobenyloxycarbonyl). An excess of a desired monomer tobe coupled may be jetted on the reaction support, followed by jetting ofa coupling reagent such as dicyclohexylcarbodiimide in a suitablesolvent such as 50% DMF in dichloromethane. Boc deprotection may then beaccomplished by jetting of trifluoroacetic acid. After completion of thedesired chain, the PNA may be cleaved from the reaction support byjetting of a mixture of trifluoromethanesulfonic acid:trifluoroaceticacid:meta-cresol (1:8:1 v:v:v) . The product is precipitated from thesolution by the addition of diethyl ether.

The methods of the present invention may be advantageously employed tosynthesize deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)(together "oligonucleotide") species by any of the several chemistriesfor solid phase DNA or RNA synthesis, including phosphite triester,(phosphoramidite) synthesis and H-phosphonate synthesis. See NucleicAcids in Chemistry and Biology, M. Blackburn and M. Gait, Eds., IRLPress 1990, at pages 112-130. In principle, the methods of the presentinvention will be useful in the practice of any iterative nucleic acidsynthetic technique. In preferred embodiments of the inventionoligonucleotides are synthesized by the phosphoramidite method.Representative solid-phase synthetic methodologies useful in the presentinvention may be found in Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066;4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat.Nos. 4,725,677 and Re. 34,069.

Media suitable for use as reaction supports in connection witholigonucleotide synthetic applications of the invention includecontrolled-pore silica glass (CPG); oxalyl-controlled pore glass (see,e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527); RAPPcopolymer highly crosslinked polystyrene, TENTAGEL Support, (see, e.g.,Wright, et al., Tetrahedron Letters 1993, 34, 3373); HLP, a high loadedpolystrene available from ABI; POROS, a polystyrene resin available fromPerceptive Biosystems, and other media known to those in the art for usein oligonucleotide synthesis as solid supports.

For phosphoramidite synthesis according to the invention, the reactionsupport is preferably functionalized with spacer groups according tomethods known in the art. Typical of such spacer groups are long chainalkylamines. The spacer groups may be keyed to preselected portions ofthe reaction support, or to the entire reaction support surface.

Keying of the initial monomeric synthon may be accomplished by jettingappropriately derivitized nucleoside monomers (having protecting groupson any exocyclic amine functionalities present) onto the reactionsupport. For example, a 5'-O-DMT-3'-O-(4-nitrophenyl) succinate monomermay be jetted onto the reaction support, keying the initial monomer tothe reaction support through the alkylamine spacer. Typically, monomericsynthons bear temporary protecting groups at appropriate nucleobase or2'-O positions. See Nucleic Acids in Chemistry and Biology, supra.

Solid phase nucleic acid synthetic techniques employ "temporary" and"permanent" protecting groups in analogous fashion to solid phasepeptide synthesis. Base labile protecting groups are used to protect theexocyclic amino groups of the heterocyclic nucleobases during thesynthesis. This type of protection is generally achieved by acylationwith acylating reagents such as benzoylchloride and isobutyrylchloride.Acid labile protecting groups are used to protect the nucleotide 5'hydroxyl during synthesis. Representative hydroxyl protecting groupscommonly used in the art may be found in Beaucage, et al., Tetrahedron1992, 48, 2223. These include the dimethoxytrityl, monomethoxy trityl,trityl, and 9-phenyl-xanthene (pixyl) groups. Dimethoxytrityl protectinggroups are widely used owing to their great acid lability, which affordsefficient removal by dilute acid (e.g. 3% trichloroacetic acid).

The first step in the iterative chain elongation cycle according to thephosphoramidite technique is the removal 5'-O-protecting group(deprotection) of the initial monomer by jetting an appropriatedeprotecting reagent onto the preselected portions of the reactionsupport. This is followed by the jetting of a rinsing reagent. Suitablereagents for deprotection include Lewis acids such as ZnBr₂, AlCl₃, BF₃and TiCl₄ in solvents such as nitromethane, tetrahydrofuran, and mixedsolvents such as nitromethane and lower alkyl alcohols, such asmethanol. Protic acids such as acetic acid, dichloroacetic acid,trifluoroacetic acid, and toluenesulfonic acid may also be used in anappropriate solvent, typically dichloromethane.

Chains are lengthened by jetting and reaction of activated5'-O-protected monomeric synthons. In the phosphoramidite technique a5'-DMTr-deoxynucleoside-3'-0-(N,N-diisopropylamino)-β-cyanoethylphosphiteis jetted onto the reaction support. Phosphoramidites of numerousnucleosides are commercially available (for example, from AppliedBiosystems Inc. and Millipore Corp.).

A mild organic acid catalyst, typically tetrazole, is jetted onto thereaction support either together with the phosphoramidite orindependently thereafter. Commonly used commercially availableactivating agents are disclosed in U.S. Pat. No. 4,725,677 and inBerner, S., Muhlegger, K., and Seliger, H., Nucleic Acids Research 1989,17:853; Dahl, B. H., Nielsen, J. and Dahl, O., Nucleic Acids Research1987, 15:1729; and Nielson, J. Marugg, J. E., Van Boom, J. H., Honnens,J., Taagaard, M. and Dahl, O., J. Chem. Research 1986, 26, all of whichare herein incorporated by reference. The coupling reaction is followedby the jetting of a rinsing solvent, typically anhydrous acetonitrile.

After rinsing, a capping reagent is jetted onto the preselected portionsof the reaction support to cap free hydroxyl species remaining due toincomplete reaction of phosphite monomers. The capping reagent, which istypically a solution of an acid anhydride, also functions to reverse anyinadvertent phosphitylation of guanine 0-6 positions.

Oxidation of the resulting phosphite triester to the correspondingphosphate triester may be accomplished by jetting oxidants known in theart to be suitable, such as a solution of alkaline Iodine.

The methods of the present invention may be employed in the synthesis ofoligonucleotides having the naturally occurring nucleobases adenine (A),thymine (T), guanine (G), cytosine (C) and uracil (U), as well asnon-naturally occurring nucleobases. Non-naturally occurring nucleobasesare molecular moieties which are known in the art to mimic the functionof naturally occurring nucleobases in their biological role ascomponents of nucleic acids. Examples of non-naturally occurringnucleobases are disclosed in, for example, Antisense Research andApplications, Crooke and Lebleu, eds., CRC Press, Boca Raton, 1993.

Further details of methods useful for preparing oligonucleotides may befound in Sekine, M., etc al., J. Org. Chem., 1979, 44, 2325; Dahl, O.,Sulfur Reports, 1991, 11, 167-192; Kresse, J., et.al., Nucleic AcidsResearch, 1975, 2, 1-9; Eckstein, F., Ann. Rev. Biochem., 1985, 54,367-402; and Yau, E. K. U.S. Pat. No. 5,210,264 entitled"S-(2,4-Dichlorobenzyl)-β-Cyanoethyl Phosphorothioate Diester".

Oligonucleotide species having a wide variety of modifications tonucleobases, sugars, or inter-sugar linkages can be prepared inaccordance with the methods of the invention, which are generallyapplicable to the synthesis of any oligomer synthesizable by solid phasetechniques. For example, the methods of the present invention may beemployed in the synthesis of S-phosphorodithioates, phosphorothioates,methyl phosphonates, phosphoramidates, phosphorotriesters,thiophosphotriesters, thiophosphoramidates, methylphosphonothioates, andcyclic phosphorothioates, phosphorothioates, and phosphorodithioates.These modifications are disclosed as set forth in Antisense Research andApplications, supra, and in U.S. patent applications assigned to acommon assignee hereof, entitled "Backbone Modified OligonucleotideAnalogs," Ser. No. 703,619 and "Heteroatomic Oligonucleotide Linkages,"Ser. No. 903,160, the disclosures of which are incorporated herein byreference.

The polymers produced according to the methods of the invention may becomposed of more than one type of monomeric subunit (e.g., amino acids,peptide nucleic acids, nucleotides, sugars, etc ) and may possess morethan one type of inter-subunit linkage. Illustrative polymers producedaccording to the methods of the invention include peptides, peptoids(N-alkylated glycines), α-polyesters, polythioamides, N-hydroxy aminoacids, β-esters, polysulfonamides, N-alkylates polysulfonamides,sulfonamides, polyureas, urethanes, peptide nucleic acids, nucleotide,polysaccharides, polycarbonates, oligonucleotide, oligonucleosides andthe like and chimeric molecules that contain one or more of thesepolymers joined together as a single macro-molecule.

Libraries of monomeric analog species can also prepared by the methodsof the invention. These include benzodiazepine libraries and other suchanalog libraries including, but not limited to, antihypertensive agents,e.g. enalapril, β-blockers, e.g. proproanol; antiulcer drugs (H₂-receptor antagonists) e.g. cimetidine and ranitidine; antifungal agents(cholesterol-demethylase inhibitors, e.g. isoconazole; anxiolytics, e.g.diazepam; analgesics, e.g. aspirin, phenacetamide, and fentanyl;antibiotics, e.g. vancomycin, penicillin and cephalosporin;antiinflammatories, e.g. cortisone; contractives, e.g. progestins;abortifacients, e.g. RU-456; antihistamines, e.g. chlorphenamine;antitussives, e.g. codeine; sedatives, e.g. barbitol and well as manyothers that will be suggested by this disclosure. Illustrative are thebenzodiazepine and the hetero-Diels-Alder libraries as described inpublished PCT application WO 94/08051; the benzodiazepine andprostaglandins described in U.S. Pat. No. 5,288,514; the dipeptides,hydantoins, benzodiazepins, quinolones, keto-ureas,benzamido-5-oxopentanoic acids, diketopeperazines, 2H-pyranones, N-arylpiperazines, benzoisothiazolones, spirosuccimides, pilocarpine analogs,benzopyrans, pyrimidinediones and tepoxalin analogs described in U.S.Pat. No. 5,324,483.

EXAMPLES Example 1 Synthesis of Library of Compounds Incorporating P^(V)Linkages Reagents

Abbreviations and Definitions:

DCM dichloromethane

Deblocking agent 3% trichloroacetic acid in DCM

ACN acetonitrile

1st Capping Sol. N-methylimidazole

2nd Capping Sol. acetic anhydride

Oxidizer Iodine for (P=O) or Beaucage reagent for (P=S)

Activator 1-H-tetrazole

Amidite One of various reactant species activated as a phosphoramidite

Position set A predetermined set of positions where a reaction is totake place--may vary for one to all positions wherein jetting head istargeted to dispense reagents

All positions Every position where jetting head is targeted to dispensereagents

Support:

Membrane Carrier: Membrane strips are positioned on the base plate andgasket and overlaid with the top plate of a 96 well, 6-mm dot GibcoBRL"The Convertible Filtration Manifold System" device from LifeTechnologies, Gaithersburg, Md. The "well" vacuum line of the carrier ismodified to include an electromechanical in line off-on value forcontrol of the vacuum below the membranes. The membrane carrier islocated vertically below a droplet generator for jetting reagentdroplets. Actuation of the in line vacuum value is via the controller32.

Cycle:

1. Deliver deblocking agent to all positions on the support

2. Wait 30 seconds

3. Remove by vacuum

4. Deliver ACN to all positions and remove by vacuum

5. Repeat step 4 five times

6. Deliver amidite A to first designated position set

7. Deliver activator to same designated position set

8. Wait 1-5 minutes as required for current amidite being used

9. Remove amidite and activator by vacuum

10. Deliver ACN to same designated position set

11. Remove by vacuum

12. Repeat steps 6-11 for further amidite B to second designatedposition set, amidite C to third designated position set, etc. forfurther amidites at designated position sets

13. Deliver ACN to all positions and remove by vacuum

14. Deliver 1st and 2nd capping solutions to all positions

15. Wait 30 seconds

16. Remove by vacuum

17. Deliver ACN to all positions and remove by vacuum

18. Repeat step 15 five times

19. Deliver oxidizer to all positions

20. Wait 30 seconds

21. Deliver ACN to all positions and remove by vacuum

22. Repeat step 19 five times

The cycle is repeated n times to achieve oligomer n residues long.

Example 2 Synthesis of Library of Compounds Incorporating Amide Linkages-BOC chemistry

Regents, Abbreviations and Definitions:

DCM dichloromethane

DMF dimethylforamide

TFA trifluoroacetic acid

HATU O-(7-azabenzotriazol-1-yl)-1,1,2,2-tetramethyl uroniumhexafluorophosphate

MDCHA N-methyldicyclohexylamine

Deblocking agent TFA/m-cresol, 95/5, v/v

Cap acetic anhydride/collidine/DMF, 5/6/89, v/v/v

Pyridine Pyridine/DMF, 5/95, v/v

Piperdine Piperdine/DMF, 5/95, v,v

Activator A 0.18M HATU in DMF, 855 mg plus 12 mL DMF

Activator B 0.3M HATU in DMF, 570 mg plus 4.7 mL DMF

Position set A predetermined set of positions where a reaction is totake place--may vary for one to all positions wherein jetting head istargeted to dispense reagents

All positions Every position where jetting head is targeted to dispensereagents

Monomers One of various reactant species capable of being linkedtogether via amide linkages

Support:

Membrane Carrier: Membrane are strips positioned on the base plate andgasket and overlaid with the top plate of a 96 well, 6-mm dot GibcoBRL"The Convertible Filtration Manifold System" device from LifeTechnologies, Gaithersburg, Md. The "well" vacuum line of the carrier ismodified to include an electromechanical in line off-on value forcontrol of the vacuum below the membranes. The membrane carrier islocated vertically below with the jetting head. Actuation of the in linevacuum value is via the controller 32.

Cycle:

1. Deliver DMF/DCM to all positions and remove by vacuum

2. Wait 10 seconds

3. Deliver TFA to all positions

4. Wait 10 seconds

5. Deliver TFA to all positions

6. Wait 180 seconds

7. Deliver TFA to all positions

8. Wait 180 seconds

9. Remove by vacuum

10. Deliver DMF/DCM wash and remove by vacuum

11. Deliver pyridine to all positions

12. Deliver DMF/DCM to all positions

13. Deliver monomer A and HATU activator to first designated positionset

14. Wait 840 seconds

15. Deliver DMF/DCM wash to first designated position set

16. Remove by vacuum

17. Repeat, in parallel, steps 13-16 for further monomer B to seconddesigned position set, etc. for further monomers at designated positionsets

18. Deliver DMF/DCM wash to all positions

19. Deliver cap to all positions

20. Wait 300 seconds

21. Deliver piperidine/DMF to all positions

22. Wait 60 seconds

23. Remove by vacuum

24. Deliver DMF/DCM wash to all positions and remove by vacuum

The cycle is repeated n times to achieve oligomer n-residues long.

Optional cleavage of library compounds from support is effected using acleavage cocktail of m-cresol/thioanisole/TFMSA/TFA, 1/1/2/6, v/v/v/v.The membrane is treated with the cleavage cocktail for one hour followedby removal from the membrane by vacuum into individual wells of anappropriate matrix collection plate.

Example 3 Synthesis of Library of Compounds Incorporating Amide Linkages-FMOC chemistry

Regents, Abbreviations and Definitions:

DCM dichloromethane

DMF dimethylforamide

TFA trifluoroacetic acid

HATU O-(7-azabenzotriazol-1-yl)-1,1,2,2-tetramethyluroniumhexafluorophosphate

MDCHA N-methyldicyclohexylamine

Deblocking agent 20% piperdine in DMF

Cap acetic anhydride/collidine/DMF, 5/6/89, v/v/v

Pyridine Pyridine/DMF, 5/95, v/v

Piperdine Piperdine/DMF, 5/95, v,v

Activator A 0.18M HATU in DMF, 855 mg plus 12 Ml DMF

Activator B 0.3M HATU in DMF, 570 mg plus 4.7 Ml DMF

Position set A predetermined set of positions where a reaction is totake place--may vary for one to all positions wherein jetting head istargeted to dispense reagents

All positions Every position where jetting head is targeted to dispensereagents

Monomers One of various reactant species capable of being linkedtogether via amide linkages

Support:

Membrane Carrier: Membrane are strips positioned on the base plate andgasket and overlaid with the top plate of a 96 well, 6-mm dot GibcoBRL"The Convertible Filtration Manifold System" device from LifeTechnologies, Gaithersburg, Md. The "well" vacuum line of the carrier ismodified to include an electromechanical in line off-on value forcontrol of the vacuum below the membranes. The membrane carrier islocated vertically below with the jetting head. Actuation of the in linevacuum value is via the controller 32.

Cycle:

1. Deliver DMF/DCM to all positions and remove by vacuum

2. Wait 10 seconds

3. Deliver TFA to all positions

4. Wait 10 seconds

5. Deliver TFA to all positions

6. Wait 180 seconds

7. Deliver TFA to all positions

8. Wait 180 seconds

9. Remove by vacuum

10. Deliver DMF/DCM wash and remove by vacuum

11. Deliver pyridine to all positions

12. Deliver DMF/DCM to all positions

13. Deliver monomer A and HATU activator to first designated positionset

14. Wait 840 seconds

15. Deliver DMF/DCM wash to first designated position set

16. Remove by vacuum

17. Repeat, in parallel, steps 13-16 for further monomer B to seconddesigned position set, etc. for further monomers at designated positionsets

18. Deliver DMF/DCM wash to all positions

19. Deliver cap to all positions

20. Wait 300 seconds

21. Deliver piperidine/DMF to all positions

22. Wait 60 seconds

23. Remove by vacuum

24. Deliver DMF/DCM wash to all positions and remove by vacuum

25. Repeat cycle by starting at step 13

The cycle is repeated n times from step 13 to achieve oligomern-residues long.

Optional cleavage of library compounds from support is effected using acleavage cocktail of 95% trifluoroacetic acid containing 5% scavenger.The membrane is treated with the cleavage cocktail for one hour followedby removal from the membrane by vacuum into individual wells of thematrix collection plate.

Example 4 Synthesis of Library of Compounds Incorporating HydroxylamineLinkages

Regents, Abbreviations and Definitions:

DCM dichloromethane

Deblocking agent p3% N-methyl hydrazine in DCM:methanol (9:1, v:v)

GAA Glacial Acetic Acid

Reducing reagent NaCNBH₃

Alkylating reagent 20% Formaldehyde

TBAF Tetrabutylammonium fluoride

THF Tetrahydrofuran

Intermediate Monomer One of various5'--O-phthalimido-3'--C-aldehydo-3'-deoxynucleosides

First Monomer One of various5'--O-phthalimido-3'--O-(succinyl)nucleosides

Final Monomer One of various5'-t-butyldiphenylsilyl-3'-aldehyde-3'-deoxy nucleosides

Membrane activator Pentachlorophenol

Position set A predetermined set of positions where a reaction is totake place--may vary for one to all positions wherein jetting head istargeted to dispense reagents

All positions Every position where jetting head is targeted to dispensereagents

Support:

Membrane Carrier: Membrane strips are positioned on the base plate andgasket and overlaid with the top plate of a 96 well, 6-mm dot GibcoBRL"The Convertible Filtration Manifold System" device from LifeTechnologies, Gaithersburg, Md. The "well" vacuum line of the carrier ismodified to include an electromechanical in line off-on value forcontrol of the vacuum below the membranes. The membrane carrier islocated vertically below with the jetting head. Actuation of the in linevacuum value is via the controller 32.

Cycle:

1. Deliver DCM solution of First Monomer A and Membrane activator to afirst designated position set

2. Wait 15 seconds

3. Wash with DCM and remove by vacuum

4. Repeat steps 1-3 for further First Monomer B to second designatedposition set, etc for further First Monomers to designated position sets

5. Deliver Deblocking agent to a first designated position set

6. Wait 120 seconds

7. Wash with DCM for 240 seconds and remove by vacuum

8. Deliver a DCM solution of a selected Intermediate monomer and GAA tothis first designated position set

9. Wait 60 seconds

10. Wash with DCM and remove by vacuum

11. Repeat steps 5 to 10 for further Intermediate monomer B to seconddesignated position set, etc for further Intermediate monomers todesignated position sets

12. Repeat steps 5 to 11 n-2 times for oligomer n-residues long

13. Deliver a DCM solution of a first Final monomer to a firstdesignated position set

14. Wait 60 seconds

15. Wash with DCM and remove by vacuum

16. Repeat steps 13 to 15 for further Final monomer B to seconddesignated position set, etc for further Intermediate monomers todesignated position sets

17. Wash with DCM and remove by vacuum

18. Deliver mixture of Reducing agent, Alkylating reagent and GAA to allpositions

19. Wait 180 seconds

20. Wash with DCM and remove by vacuum

21. Deliver TBAF in THF at all positions to de-block all Finalnucleosides

22. Wait 30 seconds

23. Wash with DCM and remove by vacuum

The oligomers are removed from membrane by treating with 30% ammoniumhydroxide.

Example 5 Synthesis of Peptide Nucleic Acid Library

A library of peptide nucleic acids, wherein individual peptide nucleicacid are 6-mers, is synthesizer utilizing the protocol of Example 2.Four nucleobase monomers are used in construction the library. Themonomers incorporate normal nucleobases attached to the peptide nucleicacid backbone wherein A is an adenine peptide nucleic acid monomer, G isa guanine peptide nucleic acid monomer, C is a cytosine nucleic acidmonomer and T is a thymine nucleic acid monomer. The monomers are usedat a 0.2M concentration. BOC (terminal amine groups) and Z (nucleobases)protection is utilized. The monomers and HATU are purchased fromMillipore Corp., Bedford Mass. The membrane is crosslinked PEPS filmthat is aminomethylated. The aminomethylated film is further modifiedwith a BOC-Try(BrZ)Pam linder from Star Chemicals coupled as a preformedHOBt ester at about 0.15 mmol amino groups per gram of film with theremaining amino groups capped by acetylation with acetic anhydride.

    ______________________________________    Monomers    (0.2 M monomer, 0.2 M MDCHA and 0.3 M collidine)    Monomer           Weight (mg)                     MDCHA (μl)                                collidine (μL)                                         Solvent (mL)    ______________________________________    A      528       214        198      4.3    G      544       214        198      4.3    T      384       214        198      4.3    C      504       214        198      4.3    ______________________________________

The monomers are appropriately solubilized in either DMF orN-methylpyrrolidinone.

Example 6 Synthesis of Peptide Library

A library of 8 mer peptides is synthesized utilizing the protocol ofExample 3. The complexity of the library is based upon the 8 aminoacids, alanine (A), arginine (R), glycine (G), leucine (L), lysine (K),serine (S), tyrosine (Y) and histidine (H) . The monomer are used at0.3M concentration. FMOC protection is utilized. The monomers and HATUare purchased from Millipore Corp., Bedford Mass. The membrane is apolypropylene coated with polyhydroxypropylacrylate as described inposter #T81 and accompanying poster #T80 of the 1989 Protein Societymeeting, San Diego, Calif. Standard peptide oligomerization protocol arefollowed.

Example 7 Synthesis of Phosphate Oligonucleotide Library

A library of 10 mer oligonucleotides is synthesized utilizing theprotocol of Example 1 with iodine as the oxidizer. The membrane used isa "Nucleic Acid Membrane Support", (from Millipore Corp.--apolyvinylidene difluoride polymeric membrane derivitized with diaminepropane). Monomer are standard deoxyribonucleotides purchased fromMillipore, Corp., Bedford, Mass. or Glen Research, Sterling, Va.

Example 8 Synthesis of Phosphorothioate Oligonucleotide Library

A library of 10 mer oligonucleotides is synthesized utilizing theprotocol of Example 1 with Beaucage reagent as the oxidizer. Themembrane used is a "Nucleic Acid Membrane Support", (from MilliporeCorp.--a polyvinylidene difluoride polymeric membrane derivatized withdiamine propane). Monomer are standard deoxyribonucleotides purchasedfrom Millipore, Corp., Bedford, Mass. or Glen Research, Sterling, Va.

Example 9 Synthesis of Oligoribonucleotide Library

A library of 6 mer oligoribonucleotides is synthesized utilizing theprotocol of Example 1 with iodine as the oxidizer. The membrane used isa "Nucleic Acid Membrane Support", (from Millipore Corp.--apolyvinylidene difluoride polymeric membrane derivatized with diaminepropane). Monomer are standard deoxyribonucleotides available fromeither Millipore, Corp., Bedford, Mass. or Glen Research, Sterling, Va.

Example 10 Synthesis of Chimeric PhosphateOligoribonucleotide-Phosphorothioate Oligodeoxyribonucleotide Library

A library of 8 mer oligonucleotides having an internal section of 4consecutive phosphorothioate deoxyribonucleotides flanked by 2 merribonucleotides is synthesized utilizing the protocol of Example 1 witheither iodine or Beaucage reagent used as the oxidizer as appropriate.The ribonucleotides are selected as 2'-O-methylribonucleotides. Thenucleobases are A, C, G and T for the deoxy portion of the chimera andA, C, G and U for the ribo portions. The membrane used is a "NucleicAcid Membrane Support", (from Millipore Corp.--a polyvinylidenedifluoride polymeric membrane derivatized with diamine propane).Monomers are standard deoxyribonucleotides or 2'-O-methylribonucleotides available for either Millipore, Corp., Bedford, Mass. orGlen Research, Sterling, Va.

Example 11 Synthesis of Oligomeric Library IncorporatingPropane-1,2-diol Monomeric Units Connected Via Phosphate Linkers

A library of 8 mer oligomers based on a propane-1,2-diol backbone linkedvia phosphate linkages and that incorporates 4 nucleobase for diversityis synthesized utilizing the protocol of Example 1 with iodine as theoxidizer. The membrane used is a "Nucleic Acid Membrane Support", (fromMillipore Corp.--a polyvinylidene difluoride polymeric membranederivatized with diamine propane). Monomer are 1{{N-{2-9-(N2-isobutyroyl)guanine!acetyl}-amino}}-3-O-dimethyoxytritylmethyl-1-amino-2-O-(N,N-disiopropylamino)-2-cyanoethoxyphosphite!propane, 1{{N-{2-9-(N6-benzoly)adenine!acetyl}amino}}-3-O-dimethyoxytritylmethyl-1-amino-2-O-(N,N-disiopropylamino)-2-cyanoethoxyphosphite!propane, 1{{N-{2-9-(N4-benzoly)cytosine!acetyl}amino}}-3-O-dimethyoxytritylmethyl-1-amino-2-O-(N,N-disiopropylamino)-2-cyanoethoxyphosphite!-propane and 1{N-{2-1-thymidine)acetylamino}-3-O-dimethyoxytritylmethyl-1-amino-2-O-(N,N-disiopropylamino)-2-cyanoethoxyphosphite!propane.

Example 12 Synthesis of Oligomeric Library Incorporating3-Hydroxypyrrolidine Monomeric Units Connected Via Phosphate Linkers

A library of 6 mer oligomers base on a hydroxypyrrolidine backbonelinked via phosphate linkages and that incorporates 6 diversity moietieshaving various characteristics is synthesized utilizing the protocol ofExample 1 with iodine as the oxidizer. The membrane used is a "NucleicAcid Membrane Support", (from Millipore Corp.--a polyvinylidenedifluoride polymeric membrane derivatized with diamine propane) .Monomer are N¹ -palmitoyl-5-dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-diisopropylamino)-2-cyanoethoxyphosphite, N¹-phenylacetyl-5-dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-diisopropylamino)-2-cyanoethoxyphosphite, N¹-(fluorenylmethylsuccinoyl)-5-dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-diisopropylamino)-2-cyanoethoxyphosphite, N¹-(N-Fmoc-3-aminopropionoyl)-5-dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-diisopropylamino)-2-cyanoethoxyphosphite and N¹-(N-imidazolyl)-5-dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-diisopropylamino)2-cyanoethoxyphosphite to give a six folddiversity incorporated in the oligomers.

Example 13 Synthesis of Oligonucleoside Library Incorporating NucleosideMonomeric Units Connected Via Hydroxylamine Linkages

A library of 6 mer oligonucleosides linked via methylenehydroxylaminolinkages and that incorporates 4 nucleobases as diversity moieties issynthesized utilizing the protocol of Example 4. The membrane used is a"Nucleic Acid Membrane Support", (from Millipore Corp.--a polyvinylidenedifluoride polymeric membrane derivatized with diamine propane).Monomers areN4-benozyl-3'-deoxy-3'-C-formyl-5'-O-phthalimido-5-methylcytosine,N6-benozyl-3'-deoxy-3'-C-formyl-5'-O-phthalimido-adenosine,N2-isobutryl-3'-deoxy-3'-C-formyl-5'-O-phthalimido-guanosine and3'-deoxy-3'-C-formyl-5'-O-phthalimido-thymidine.

What is claimed is:
 1. A method for chemical synthesis comprising:a.providing a reaction support having a first and second surfaces, saidfirst surface having a plurality of selected sites for chemicalsynthesis, said second surface located adjacent to a collection platehaving a plurality of collection wells such that one or more of saidselected sites for chemical synthesis are associated with a collectionwell throughout said synthesis; b. providing one or more dropletgenerators actuated through a piezoelectric device, each connected toone or more chemical reservoirs for jetting chemical reagents; c.delivering by way of said droplet generator or generators to saidselected sites on the first surface of said reaction support a pluralityof chemical reagents, the chemical nature, amount and sequence of whichis predetermined and under the control of a control means to effectsynthesis of at least one chemical species; d. upon complete synthesisof said chemical species on said reaction support at selected sitestransporting said chemical species through the reaction support to saidcollection wells associated with said selected sites of said reactionsupport such that at least one chemical species is collected in saidcollection well or wells.
 2. The method of claim 1 further comprisingchallenging said chemical species in an assay.
 3. The method of claim 1wherein said delivery of chemical reagents is performed iteratively toeffect the synthesis of an oligomer.
 4. The method of claim 3 whereinsaid oligomer is an oligonucleotide.
 5. The method of claim 3 whereinsaid oligomer is a polypeptide.
 6. The method of claim 3 wherein saidoligomer is a peptide nucleic acid.
 7. The method of claim 1 whereinsaid jetting droplet generator is a continuous droplet generator.
 8. Themethod of claim 1 wherein said jetting droplet generator is a droplet ondemand generator.
 9. The method of claim 1 wherein said reaction supporthas a porosity such that liquid is capable of passing from said firstsurface to said second surface.
 10. The method of claim 9 wherein saidreaction support is selected from the group consisting of permeable andsemipermeable membranes, polymeric membranes, and porated membranes andglasses.
 11. The method of claim 10 wherein said reaction support iscontrol pore glass.